This is a revised renewal application for a NIMH Research Scientist Award, for years 21-25 in the Research Scientist Program. The focus is HPA axis hyperactivity in adult and adolescent major depression. The working hypothesis is that this hyperactivity is central nervous system (CNS)- driven, as a result of increased CNS cholinergic neurotransmission, with normal adaptive physiology of the axis itself (no qualitative defects). To elucidate this, studies are being undertaken at several levels of the HPA axis in major depressives and individually matched normal controls, including development of a low-dose CNS cholinergic stimulation test devoid of confounding side effects, and use of ovine CRH (OCRH) stimulation to assess corticotroph downregulation, low-dose ACTH1.24 stimulation to assess adrenal gland sensitivity, and magnetic resonance (MR) imaging of the adrenal for size determination. Plasma ACTH, cortisol, and oCRH will be measured, as well as bioactive ACTH. Future planned experiments include: At the CNS level, stimulation of the HPA axis following pharmacologic stimulation of central cholinergic neurotransmission, to test the hypothesis of increased central cholinergic neurotransmission underlying HPA axis hyperactivity in major depression. At the pituitary level, oCRH challenge studies with and without metyrapone pretreatment to suppress cortisol, to clarify the contribution of cortisol feedback to corticotroph downregulation. At the adrenal level, additional low-dose ACTH1.24 challenge studies to find the optimal dose for discriminating differences in adrenal sensitivity between depressives and controls, and MR imaging of the adrenals for volume determination. A further hypothesis is that HPA axis hyperactivity in depression is state-dependent, and that following treatment, the hormone measures will return to normal in a consistent manner. Thus, patients are being studied a second time when they are in remission and off medication for at least one month. Planned for research development are studies with functional neuroimaging. specifically SPECT. This neuroimaging technique is useful for determining regional cerebral blood flow (rCBF) and uptake of compounds like HMPAO, which also reflects blood-brain barrier, and possibly cell membrane, permeability. The hypothesis is that depressives will show decreases in rCBF and HMPAO uptake especially in the orbital frontal cortex. Work with specific cholinergic receptor ligands for SPECT and PET also is planned.